A 40-color immunophenotyping panel for a spectral flow cytometer that targets T, B, NK and NKT cells, neutrophils, monocytes, basophils, dendritic cells and innate lymphoid cells. It incorporates markers previously reported in OMIP-109, with some minor updates.
Whole blood is labeled with the 40-color panel and acquired on a spectral flow cytometer, yielding high-resolution data on dozens of immune-cell populations at once.
A few milliliters per sample
39 antibodies plus a viability dye
Spectral emissions of every dye
Multiparametric cytometry data
The preliminary panel incorporates markers previously reported in OMIP-109, with some minor updates. A 30-color panel has already been established and tested on normal samples.
A viability dye discriminates live from dead cells, and tetramer staining will be employed to track antigen-specific T cells.
High-resolution data depend on factors such as reagent concentration. The panel is optimized on whole blood and peripheral blood mononuclear cells (PBMCs) from healthy donors sourced from a local blood bank.
The panel provides insight into the expression of fundamental markers on immune cells and into the cells’ differentiation and activation status. Select a marker to see its role and the reference phenotypes that express it.
Select a marker to see its category, role and the populations that express it.
The 40-color staining covers lymphoid and myeloid lineages. Figure 3 of the proposal lists the targeted populations; the research portal defines 40 analysis populations based on that list — an adaptation of Figure 3, not a one-to-one copy.
30 mutually exclusive leaf populations add up to 100% of live CD45⁺ leukocytes — the compositional data analyzed in centered log-ratio space and by optimal transport. 10 overlapping functional subsets are reported as a percentage of their parent. 23 populations are flagged as rare.
Reported changes in AD refer to proposal Figure 1 (literature). Gating phenotypes are the portal's reference definitions.
Blood samples are obtained from healthy donors in the local blood bank. The optimized staining panel is validated according to recommendations from CLSI H62; if the criteria are met, the panel is then used to immunophenotype patients with AD, alongside relevant controls.
| Validation Parameter | Samples | Acceptance Criteria |
|---|---|---|
| Intra-Assay Precision |
|
% CV ≤ 25/35 |
| Inter-Assay Precision |
|
% CV ≤ 25/35 |
| Inter-Operator Variability |
|
% Difference ≤ 20 |
| Sample Stability |
|
% Change from baseline ≤ 20 |
| Biological variability |
|
To be reported as found |
Agreement of replicate measurements of the same sample within an analytical run.
Agreement of results for the same donors across separate analytical runs.
Agreement between results produced by different operators.
Change from baseline when samples are analyzed after 36 and 48 hours.
Natural differences between healthy donors, reported as found.
PhenArI has standard operating procedures that align with CLSI H62 recommendations on daily usage and performance monitoring of instruments and on new reagent lots for flow cytometry applications.
Instrument performance is monitored every day of use.
Each analytical run includes at least one quality-control sample.
New reagent lots for flow cytometry applications are covered by the same CLSI H62-aligned SOPs.
Data are excluded if significant deviations in daily quality control are observed.
Once the panel passes CLSI H62 validation, Aim 2 follows AD patients on lecanemab or donanemab through their treatment cycle.
